Katharina M. Detjen

Activated signal transducer and activator of transcription 3 (STAT3) supports the malignant phenotype of human pancreatic cancer

BACKGROUND & AIMS Constitutive activation of signal transducer and activator of transcription 3 (STAT3) has been implicated in regulation of growth and malignant transformation. We therefore analyzed the expression and biologic significance of STAT3 in human pancreatic cancer cells. METHODS Expression and activation of STAT3 were investigated by immunohistochemistry and immunoblotting. Functional inactivation of STAT3 was achieved by stable transfection of dominant-negative STAT3 constructs in 2 pancreatic cancer cell lines and confirmed by electrophoretic mobility shift assay and immunoblotting. Cell proliferation and tumorigenicity were evaluated by cell counting, colony formation in soft agar, and xenotransplantation in nude mice. STAT3-dependent cell cycle distribution was monitored by flow cytometry, immunoprecipitation, immunoblotting, and histone H1 and GST-Rb kinase assays. RESULTS Compared with nontransformed human pancreas, activated STAT3 is overexpressed in ductal carcinoma cells but not in ducts from chronic pancreatitis. Constitutive activation was also observed in all human pancreatic cancer cell lines examined. Functional inactivation of STAT3 resulted in significant inhibition of anchorage-dependent and -independent proliferation in vitro and reduced tumor growth in vivo. Cell cycle analysis showed a delay of G(1)/S-phase progression due to inhibition of cyclin-dependent kinase 2 activity based on increased expression of p21(WAF1) in vitro and in vivo. Blocking of the STAT3 upstream activator Janus kinase 2 by tyrphostin also resulted in growth arrest because of delayed G(1)/S-phase progression and increased expression of p21(WAF1). CONCLUSIONS On malignant transformation, activated STAT3 promotes cellular proliferation by acceleration of G(1)/S-phase progression and thereby contributes to the malignant phenotype of human pancreatic cancer.

Galectin-1 interacts with the {alpha}5{beta}1 fibronectin receptor to restrict carcinoma cell growth via induction of p21 and p27.

Surface binding of galectin family members has the potential to link distinct glycan structures to growth regulation. Therefore, we addressed the antiproliferative potential of galectin-1 (Gal-1) in a panel of carcinoma cell lines. We discovered growth inhibition by Gal-1 in epithelial tumor cell lines from different origins and provide evidence that this effect requires functional interaction with the α5β1 integrin. Antiproliferative effects result from inhibition of the Ras-MEK-ERK pathway and consecutive transcriptional induction of p27. We have further identified two Sp1-binding sites in the p27 promoter as crucial for Gal-1 responsiveness. Inhibition of the Ras-MEK-ERK cascade by Gal-1 increased Sp1 transactivation and DNA binding due to reduced threonine phosphorylation of Sp1. Furthermore, Gal-1 induced p21 transcription and selectively increased p27 protein stability. Gal-1-mediated accumulation of p27 and p21 inhibited cyclin-dependent kinase 2 activity and ultimately resulted in G1 cell cycle arrest and growth inhibition. These data define a novel mechanism whereby Gal-1 regulates epithelial tumor cell homeostasis via carbohydrate-dependent interaction with the α5β1 integrin.

Hypoxia-Inducible Factor 1α Mediates Anoikis Resistance via Suppression of α5 Integrin

The transcription factor hypoxia-inducible factor 1 (HIF-1) alpha is abundantly expressed in the majority of human carcinomas and their metastases. HIF-1alpha controls central metastasis-associated pathways such as glycolysis, angiogenesis, and invasion. Functional inhibition of HIF-1alpha leads to impaired metastasis formation in murine tumor models. However, the precise molecular mechanisms underlying the metastasis-promoting role of HIF-1alpha have not been fully characterized. The ability of transformed epithelial cells to initiate the metastatic cascade relies on their ability to escape anoikis, a default program of apoptosis induction following loss of integrin anchoring to the extracellular matrix. Therefore, we addressed the function of HIF-1alpha in anoikis resistance and anchorage-independent growth. Inhibition of HIF-1alpha via RNA interference resulted in up-regulation of alpha5 integrin on the cell surface of human gastric cancer cells, whereas other integrins remained unaffected. Integrin alpha5 induction occurred at the level of transcription and was dependent on elevated intracellular superoxide in HIF-1alpha-knockdown cells. HIF-1alpha-deficient cells displayed significantly increased anoikis susceptibility due to up-regulated alpha5 integrin. Finally, colony formation in soft agar was shown to be dependent on HIF-1alpha as HIF-1alpha-deficient cells displayed a 70% reduction in anchorage-independent proliferation. Results obtained by RNA interference could be entirely confirmed by application of the pharmacologic HIF-1alpha-inhibitor 2-methoxyestradiol. Hence, our data argue for a pivotal role for HIF-1alpha in anoikis control via suppression of alpha5 integrin. HIF-1alpha-inhibiting drugs might therefore offer an innovative strategy for antimetastatic cancer therapy.

Molecular mechanism of interferon alfa-mediated growth inhibition in human neuroendocrine tumor cells.

BACKGROUND & AIMS Although human neuroendocrine tumors respond to interferon (IFN)-alpha treatment in vivo, the underlying mechanisms of growth inhibition are poorly understood. To characterize the antiproliferative effects at a molecular level, we explored the growth-regulatory action of IFN-alpha in the human neuroendocrine tumor cell lines BON and QGP1. METHODS IFN-alpha receptor expression and signal transduction were examined by reverse-transcription polymerase chain reaction, immunoblotting, subcellular fractionation, and transactivation assays. Growth regulation was evaluated by cell numbers, soft agar assays, and cell cycle analysis using flow cytometry. Expression and activity of cell cycle-regulatory molecules were determined by immunoblotting and histone H1-kinase assays. RESULTS Both cell lines expressed IFN-alpha receptor mRNA transcripts. Ligand binding initiated phosphorylation of Jak kinases and Stat transcription factors, resulting in Stat activation, nuclear translocation, and transcription from an ISRE-reporter construct. Prolonged IFN-alpha treatment dose-dependently inhibited both anchorage-dependent and -independent growth. Cell cycle analysis of IFN-alpha-treated, unsynchronized cultures revealed an increased S-phase population, which was further substantiated in G(1) synchronized QGP1 cells. IFN-alpha-treated cells entered S phase in parallel to control cultures, but their progress into G(2)/M phase was delayed. Both cellular cyclin B levels and CDC 2 activity were substantially reduced. The extent and time course of this reduction corresponded to the observed S-phase accumulation. CONCLUSIONS IFN-alpha directly inhibits growth of human neuroendocrine tumor cells by specifically delaying progression through S phase and into G(2)/M. These cell cycle changes are associated with inhibition of cyclin B expression, resulting in reduced CDC2 activity.

Downregulation of p21waf/cip-1 mediates apoptosis of human hepatocellular carcinoma cells in response to interferon-γ

There is no effective treatment for advanced hepatocellular carcinoma (HCC). We therefore explored the molecular mechanisms of interferon-gamma (IFN-gamma)-mediated growth regulation in human HCC cell lines. IFN-gamma receptor expression, signal transduction, and regulation of effectors were examined by RT-PCR, immunoprecipitation, immunoblotting, and reporter gene assays. Growth and apoptosis were determined based on cell numbers, cell cycle analyses, kinase assays, DNA fragmentation, and PARP cleavage. HCC cell lines express functionally intact IFN-gamma receptors and downstream effectors. IFN-gamma profoundly inhibited growth of HCC cells via two different mechanisms: inhibition of G1 cell cycle progression and induction of apoptosis. Analyses in SK-Hep-1 cells revealed a deficient cyclin D induction in IFN-gamma-treated cells, resulting in reduced activity of CDK4 and CDK2 kinases and pRB hypophosphorylation. In contrast, apoptosis prevailed in IFN-gamma-treated HepG2 cultures. A survey of apoptosis relevant IFN-gamma effectors including IRF-1, caspase-1, caspase-3, and p21(waf/cip-1) documented a dramatic transcriptional downregulation of p21(waf/cip-1) exclusively in apoptosis-susceptible HepG2 cells. Reconstitution of p21(waf/cip-1) rescued HepG2 cells from IFN-gamma-induced apoptosis, indicating that p21(waf/cip-1) reduction was required for apoptosis execution. Inversely, downregulation of p21(waf/cip-1) sensitized SK-Hep-1 cells to IFN-gamma-induced apoptosis. Thus, downregulation of p21(waf/cip-1) in HCC cells functions as a novel, critical determinant of alternative growth inhibitory pathways in response to IFN-gamma.

Nonsteroidal anti-inflammatory drugs inhibit growth of human neuroendocrine tumor cells via G1 cell-cycle arrest.

Therapeutic options to inhibit growth of human NETs of the GEP system are limited. Since NSAIDs might provide an antiproliferative treatment alternative with acceptable toxicity, we examined the effects of different NSAIDs on growth and survival in a representative set of human GEP NET cell lines. Growth and apoptosis were determined based on cell numbers, cell‐cycle analyses, kinase assays, DNA fragmentation and PARP cleavage. Expression of COX and cell cycle–regulatory molecules was examined by immunoblotting and reporter gene assays. Depending on the drug and cell line investigated, NSAID treatment resulted in profound growth inhibition of GEP NET cells. Growth‐inhibitory effects were achieved with either COX‐2 selective (NS398) or unselective (indomethacin, sulindac) compounds. Cell‐cycle analyses documented a G1 arrest in NSAID‐treated GEP NET populations. In addition, 100 μM sulindac or indomethacin induced apoptosis. All 3 COX inhibitors prevented CDK‐2 activation. In parallel to the NSAID‐mediated reduction of CDK‐2 activity, p21cip‐1 promoter activity and cellular p21cip‐1 levels increased and p21cip‐1 was sequestered into CDK‐2 complexes. Thus, the G1 arrest likely resulted from p21cip‐1‐dependent inhibition of CDK‐2 activity. At therapeutically relevant concentrations, sulindac significantly reduced GEP NET cell numbers, whereas IFN‐α and octreotide remained ineffective. The extent of growth inhibition in GEP NETs was comparable to the antiproliferative effects of sulindac in established NSAID‐sensitive cell models. NSAIDs acted as potent antiproliferative agents in GEP NET cells via G1 cell‐cycle arrest and might therefore offer a therapeutic alternative to current treatment modalities.

The oral multitarget tumour growth inhibitor, ZK 304709, inhibits growth of pancreatic neuroendocrine tumours in an orthotopic mouse model

Background and aims: Current systemic therapies for neuroendocrine tumours (NETs) do not provide sufficient control of tumour growth. However, efficient evaluation of novel drugs is hindered by the lack of a suitable preclinical animal model. Here an orthotopic mouse model of pancreatic NET is established and used to study the action of ZK 304709, a first in class, oral multitarget tumour growth inhibitor. ZK 304709 is an inhibitor of cyclin-dependent kinases (Cdks) 1, 2, 4, 7 and 9, vascular endothelial growth factor receptor-type kinases (VEGF-RTKs) 1–3 and platelet-derived growth factor receptor-type kinase β (PDGF-RTKß). Methods: BON and QGP-1 human NET cells were used to study proliferation, survival and cell cycle distribution in vitro. For induction of orthotopic NETs, BON cells were injected into the pancreas of NMRInu/nu mice. Primary tumour growth and metastatic spread were recorded after 9 weeks, and apoptosis, microvessel density and lymphatic vessel density were determined. Results: ZK 304709 dose-dependently suppressed proliferation and colony formation of NET cells. Direct effects on NET cells were consistent with Cdk inhibition and involved G2 cell cycle arrest and apoptosis induction, which was associated with reduced expression of MCL1 (myeloid cell leukaemia sequence 1), survivin and hypoxia-inducible factor 1α (HIF1α). Apoptosis similarly occurred in vivo in ZK 304709-treated orthotopic BON tumours, resulting in a 80% reduction of primary tumour growth. In contrast, treatment with lanreotide or 5-fluorouracil and streptozotocin failed to inhibit tumour gowth. ZK 304709 also reduced tumour microvessel density, implicating antiangiogenic mechanisms. Conclusion: BON orthotopic tumours provide an informative model for preclinical drug evaluation in NETs. In this model, ZK 304709 achieved efficacious tumour growth control via induction of apoptosis and inhibition of tumour-induced angiogenesis.

Bombesin receptor gene expression in rat pancreatic acinar AR42J cells: Transcriptional regulation by glucocorticoids

BACKGROUND/AIMS This study investigated the correlation between glucocorticoid-regulated gene expression of the bombesin receptor (BR) and cellular sensitivity to bombesin stimulation in the rat pancreatic acinar cell line AR42J. METHODS BR gene expression was assessed using a cloned complementary DNA probe and radioligand binding assays. Intracellular Ca2+ mobilization was assessed by dual wavelength spectrophotometry using fura-2 in single cells. RESULTS Dexamethasone resulted in a rapid dose- and time-dependent decrease of BR messenger RNA levels with maximal inhibition to 25% +/- 2% of controls (n = 4) after 6 hours of hormone treatment. BR messenger RNA half-life was approximately 120 minutes and was not affected by dexamethasone pretreatment; nuclear run-on analysis showed a decreased transcription rate of the BR to approximately 25% of control after hormonal treatment. Radioligand binding studies showed a time-dependent decrease of specific bombesin binding to 25% +/- 8% of control after 48 hours of hormone treatment. Down-regulation of BR gene expression by dexamethasone resulted in a time- and dose-dependent decrease of intracellular Ca2+ mobilization after bombesin stimulation compared with untreated controls. CONCLUSIONS Glucocorticoids decrease BR gene transcription. The subsequent decrease in cellular BR number renders AR42J cells less sensitive for bombesin-stimulated intracellular Ca2+ mobilization.

Open-label, non-randomised, inter-individual dose escalation of ZK 304709 with the evaluation of safety, tolerability, pharmacokinetics, oral bioavailability and orientating efficacy after daily administration in patients with advanced cancer (7 d treatment and 14 d recovery)

PURPOSE The primary objectives of this study were to determine the maximum tolerated dose (MTD) and dose-limiting toxicities (DLTs) of ZK 304709, a novel multi-targeted growth inhibitor (MTGI(trade mark)), in man. Secondary end-points included safety evaluation, tolerability, pharmacokinetic profiling and assessment of response using standard and novel surrogate pharmacodynamic end-points. MATERIALS AND METHODS Patients (n=40) with advanced solid malignancies were treated with ZK 304709, administered orally once daily for 7 d with 14 d recovery. Doses were escalated in sequential cohorts of three patients with expansion to 6-7 patients should a dose-limiting toxicity occur. RESULTS ZK 304709 was safely administered up to 360mg. However, above 90mg blood concentrations increased only slightly. As this dose was not deemed likely to result in meaningful pharmacologic or clinical activity, the trial was stopped before the MTD was ascertained. It was therefore not possible to make a reliable assessment of efficacy or pharmacodynamic end-points. CONCLUSIONS Due to the lack of further increment in blood concentrations above a dose of 90mg, which was felt from previous animal studies to be unlikely to result in meaningful pharmacologic or clinical activity, this study was stopped early.

Mesenteric Fibrosis in Midgut Neuroendocrine Tumors: Functionality and Radiological Features

Background: Mesenteric fibrosis (MF) surrounding a lymph node metastasis is a known phenomenon in midgut neuroendocrine tumors (NETs) with characteristic radiological appearance. Its etiology is poorly understood as it affects some but not all midgut NET patients with lymphatic involvement. This study assessed a potential relationship of MF with carcinoid syndrome, urinary 5-hydroxyindoleacetic acid (5-HIAA), and carcinoid heart disease (CHD). Methods: A cohort of 81 patients with pathologically proven NETs with the primary site in the midgut and mesenteric lymphatic metastases on imaging were retrospectively included. Imaging characteristics of lymphatic and hepatic metastases at diagnosis (size, number, burden, and morphologic features, including presence of MF), Ki67 grading, 5-HIAA, functionality, and development of CHD were analyzed. Results: Overall, 54% of patients had MF. The presence of MF was more frequently associated with mesenteric vessel encasement (100 vs. 46% without MF; p < 0.001), presence of hepatic metastases (91 vs. 62%; p = 0.002), larger hepatic tumor burden (15 vs. 5%; p = 0.001), and functionality (86 vs. 43%; p < 0.001). Multivariate analysis revealed 5-HIAA ≥395 µmol/day (p = 0.020), age (p = 0.013), and largest lymphatic metastasis ≥24 mm (p = 0.009) as independent predictors of MF, while functionality (p = 0.098) and CHD (p = 0.070) showed a tendency towards significance. MF was associated with decreased time to development of CHD in functional midgut NETs (p = 0.043). Conclusions: We found a significant association of MF with metastatic patterns and with criteria of functionality. The association of MF with elevated 5-HIAA, and consecutively with carcinoid syndrome and potential development of CHD, suggests a linked pathophysiological mechanism, which might be similar to that of endocardial fibrosis.